1. Field of the invention
This invention relates to peroxisomal ascorbate peroxidase gene from barley, which is a novel gene induced by high temperature stress and a transgenic plant exhibiting improved thermotolerance produced by incorporating said gene.
2. Description of Related Art
Food crisis is expected to occur in the near future, caused by increase of population and alteration of environment. Therefore, in order to cope with the problem, there is a great demand on development of a agricultural technique that enables efficient production of crops. The amount of agricultural production tends to be effected by various environmental stresses. It reserves to be a serious problem in the field of agriculture. Therefore production of a plant, exhibiting resistance to various environmental stress, is needed. Especially, anathermal of the earth is a serious problem. Therefore, production of a plant having improved thermotolerance reserves to be great value.
Until now, for the production of a transgenic plant having improved thermotolerance by a genetic engineering technique, a gene encoding heat shock protein, induced by high-temperature stress, have been utilized for such purpose. Furthermore in order to attain above purpose, small amount of glycine betaine, which is a suitable solute to prevent enzyme denaturation under heat stress, was also bio-synthesized in a plant body. Moreover, it has been recognized that genes, involved in decomposing of active oxygen molecules, are important for resistance to environmental stress. Using such genes, resistance to photo-oxidation injury, resistance to salt stress or resistance to aridity stress was rendered to a plant by incorporating said gene involved in resistance to environmental stress. Despite of it, there have been no report describing on imparting of thermotolerance, achieved by incorporating genes involved in decomposition of active oxygen molecules described above.
The inventor has remarked importance of genes involved in decomposition of active oxygen molecules and challenged on imparting thermotolerance to a plant by incorporating a gene involved in decomposition of active oxygen molecules, not by conventional techniques of incorporating a gene encoding heat shock protein. That is, it is an object of this invention to develop a novel technique which can impart thermotolerance to a plant by incorporating a gene involved in defense to oxidative stress. In the concrete, it is an object of this invention to obtain a novel gene useful for such purpose and determine base sequence of the gene.
High temperature stress is one of the main factors constraining the growth and productivity of plants. Living organisms respond rapidly to high temperatures by metabolic changes that involve complex reprogramming of cellular activities. These changes must help to protect the essential structures and functions of cells against damage caused by the stress. It is known that heat shock can result in an oxidative stress, which induces genes involved in the oxidative stress defense. Under heat stress, excessive active oxygen species (AOS) such as superoxide radicals, hydrogen peroxide, and hydroxyl radicals are formed which cause oxidative damage of cell constituents.
A major hydrogen peroxide detoxifying system in plant chloroplast and cytosol is the so-called ascorbate glutathione cycle, in which APXs (ascorbate peroxidases) are the key enzymes. APX is believed to be involved in the detoxifying of photoproduced hydrogen peroxide. APX activity has been shown to increase in response to a number of stress conditions, such as drought, air pollution, high light intensity combined with chilling, UV light and deficiency in microelements.
So far, several different protein isoforms are known, those are two soluble cytosolic forms, and two chloroplast forms, one of which is stromal and the other thylakoid bound. Recently, several isoforms bound to membranes of glyoxysomes and peroxisomes were reported. Recently peroxisomal type ascorbate peroxidase was cloned from cotton (Bunkelmann and Trelease (1996) Plant Physiology 110, 589-598), Arabidopsis (Zhang et al. (1997) Plant Molecular Biology 34, 967-971) and spinach (Ishikawa et al. (1998) Plant and Cell Physiology 39, 23-34). Incidentally, novel gene according to this invention was obtained independent to known sequences described above.
Peroxisome, with glyoxysome, is an micro-organella exhibiting function, specialized to oxidative reaction utilizing molecular oxygen. These organellas described above are totally designated to microbody. The name of peroxisome is originated from production of hydrogen peroxide catalyzed by an enzyme localized at the organella. In the enzyme reaction, hydrogen atom is depleted from substrate, which is an organic compound, utilizing molecular oxygen. Moreover, catalase also exists in peroxisome. Hydrogen peroxide, generated by the reaction described above, is utilized for oxidation of compounds such as phenols, formaldehyde and alcohols by so-called xe2x80x9cperoxidative reactionxe2x80x9d. Such oxidative reaction is involved in detoxification of harmful molecules, accordingly exhibiting great importance especially in cells of liver or kidney.
Under various stress conditions, generation of hydrogen peroxide increases in microbody and the generated hydrogen peroxide diffuses into cytosol. The peroxisomal type ascorbate peroxidase of this invention is assumed to be bound outside of peroxisome membrane, operating to protect cells from injury by decomposition of hydrogen peroxide. Therefore, peroxisomal type ascorbate peroxidase of this invention would enable rapid deletion of active oxygen species generated in peroxisome before transition to cytosol, which could not be achieved by conventional ascorbate peroxidase of cytosolic form or chloroplast form.
These and other features and advantages of this invention will become apparent upon a reading of the detailed description and drawings.